Project Summary/Abstract This project focuses on the biology of telomeres, the protective elements at chromosome ends whose erosion limits the replicative lifespan of primary human cells. Telomere dysfunction has been implicated in a number of human diseases, including inherited bone marrow failure syndromes and cancer. We aim to understand how telomeres protect chromosome ends and how they are maintained. Insights into these issues resulted from our studies of shelterin, the six-subunit protein complex that binds to telomeric DNA. Shelterin is anchored on telomeres by two telomeric DNA binding proteins, TRF1 and TRF2, which promote telomere replication and protect telomeres from the DNA damage response, respectively. This proposal focuses on these two critical telomere factors. In AIM 1, we will critically test the leading model in the field that telomeres are protected through the formation of the t-loop structure by TRF2. We have previously shown that TRF2 is required for t-loop formation and our preliminary data indicates that TRF2 is sufficient to create this structure. Despite the popularity of this model, there is no direct proof that t-loops repress the DNA damage response. We are developing orthogonal (TRF2-independent) tools for t-loop formation and will test whether generation of t-loops is sufficient to repress the DNA damage response in absence of TRF2. In AIM 2, we will investigate how TRF2 generates t- loops. T-loop formation is thought to require the TRFH domain of TRF2 but how this domain acts is not clear. Our preliminary studies of the TRFH domains of TRF2 (which forms t-loops) and TRF1 (which does not) have identified features of the TRF2 TRFH domain with potential relevance to t-loop formation. These attributes will be tested for their relevance to telomere protection in vivo. In AIM 3, we will study the role of the TRF2 Myb domain in the protection of telomeres. Our preliminary data challenge the widely-held view that the TRF1/2 Myb domains only function as DNA binding modules and suggest that they have additional functions, potentially through protein-protein interactions. We will pursue our hypothesis, based on our preliminary data, that the Myb domain of TRF2 mediates a critical role in telomere protection. In AIM 4, we will investigate the function of TRF1 in telomere replication. Loss of TRF1 induces fragile telomeres, replication fork stalling, and sister telomere associations. Our preliminary data show how TRF1 uses the BLM helicase to prevent lagging-strand replication problems, most likely caused by G4 DNA. We propose to investigate how TRF1 prevents leading-strand replication problems, fork stalling, and sister telomere associations. We will expand on our preliminary data indicating that the sister telomere associations represent a novel type of telomere fusion resulting from a previously unrecognized alt-NHEJ mechanism involving stalled replication forks. This project will use innovative approaches and our established biochemical, cell biological, and molecular genetic tools to reveal new concepts in telomere biology with relevance to age-related human disease states.